Selection of aptamers against triple negative breast cancer cells using high throughput sequencing

Triple-negative breast cancer is the most aggressive subtype of invasive breast cancer with a poor prognosis and no approved targeted therapy. Hence, the identification of new and specific ligands is essential to develop novel targeted therapies. In this study, we aimed to identify new aptamers that bind to highly metastatic breast cancer MDA-MB-231 cells using the cell-SELEX technology aided by high throughput sequencing. After 8 cycles of selection, the aptamer pool was sequenced and the 25 most frequent sequences were aligned for homology within their variable core region, plotted according to their free energy and the key nucleotides possibly involved in the target binding site were analyzed. Two aptamer candidates, Apt1 and Apt2, binding specifically to the target cells with \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$K_{d}$$\end{document}Kd values of 44.3 ± 13.3 nM and 17.7 ± 2.7 nM, respectively, were further validated. The binding analysis clearly showed their specificity to MDA-MB-231 cells and suggested the targeting of cell surface receptors. Additionally, Apt2 revealed no toxicity in vitro and showed potential translational application due to its affinity to breast cancer tissue sections. Overall, the results suggest that Apt2 is a promising candidate to be used in triple-negative breast cancer treatment and/or diagnosis.

www.nature.com/scientificreports/ Breast cancer is the most commonly diagnosed cancer and the leading cause of death among women with nearly 2.1 million new cases diagnosed and 630,000 deaths in 2018 1 . Triple-negative breast cancer (TNBC) is a heterogeneous subtype of breast cancer, characterized by the absence of receptors for estrogen (ER), progesterone (PR), and epidermal growth factor receptor 2 (HER2) that represents approximately 15% of all breast cancer cases. TNBC patients have a worse prognosis comparing to other breast cancer subtypes due to its aggressive and metastatic nature, elevated rates of relapse, and a low response to current therapies [2][3][4] . Conventional chemotherapy has been the only systemic treatment option for early and advanced TNBC disease 5 . However, recently published advances have shown exciting results with poly (ADP-ribose) polymerase (PARP) inhibitors and immunotherapy agents [6][7][8] . An emerging strategy in tumor-targeted therapies consists of using specific ligands, such as aptamers that are able to bind a variety of targets including proteins, small molecules, viruses, bacteria and live cells with high affinity, specificity and selectivity [9][10][11] . Aptamers exhibit many advantages over other ligands, such as the antibodies, since they can be easily synthesized and chemically modified, are non-toxic, and possess rapid tissue penetration and low immunogenicity 12 . Aptamers are short single-stranded deoxyribonucleic acid (ssDNA) or ribonucleic acid (RNA) oligonucleotides derived from random oligonucleotide libraries through an in vitro iterative method so-called Systematic Evolution of Ligands by EXponential Enrichment (SELEX), which involves repetitive rounds of partitioning and enrichment commonly performed with purified target proteins immobilized on a solid support 13,14 . Cell-SELEX enables the generation of aptamers directed towards cell-surface molecules by using whole living cells as targets 11,15 . It allows the screening of cell-specific aptamers without any prior knowledge of molecular signatures of the target cells, besides offering the unique ability to target specific phenotypes by performing positive-negative selection cycles to find proteins only present on the target cells 16,17 . This procedure was successfully used for selecting breast cancer targeting aptamers 18,19 . For instance, Liu et al. 16 selected a ssDNA aptamer against MCF-7 breast cancer cells able to differentiate between breast cancer molecular subtypes.
However, the identification of new specific-target aptamers has been challenging mainly due to difficulties related to the characterization of the potential sequences from enriched libraries. Usually, the identification of enriched sequences is mainly performed by cloning and Sanger sequencing of the library resulting from several selection cycles 16,20 . Therefore, only sequenced clones are identified as possible targeting ligands, excluding candidate aptamers with good performances but with low copy numbers in the enriched library. To overcome this issue, specialized technologies have been incorporated into the original SELEX process such as high throughput sequencing (HTS) and bioinformatics analysis. HTS and bioinformatics combined with SELEX (HT-SELEX) enable the identification of a large number of aptamer candidates, total reads, frequencies of each unique sequence, distribution of each nucleotide in the random sequence and the rate of molecular enrichment 21 . Moreover, HT-SELEX allows identifying aptamers with high affinity and specificity at earlier selection rounds, which can greatly reduce over-selection which is time-consuming, and also avoid potential PCR artifacts in the amplification steps 22 .
In this study, we combined cell-SELEX with next-generation HTS and bioinformatics analysis to select ssDNA aptamer sequences that specifically bind to the metastatic TNBC cell line MDA-MB-231. The selected aptamers were further characterized and their affinity and specificity against the target cells were confirmed by flow cytometry and fluorescence microscopy. Moreover, the translational applicability of these aptamers was assessed using tumor tissue sections. Our results suggest that the selected aptamers are promising candidates for targeting and diagnosis applications in TNBC.

Selection of aptamers against MDA-MB-231 cells through cell-SELEX.
In the first SELEX round, a positive selection was performed. The initial ssDNA library was incubated with MDA-MB-231 cells to obtain the maximum amount of ssDNA oligonucleotides binding to the target cells. From the 2nd round onwards, the negative control cells MCF-10-2A were introduced for counter-selection to eliminate any sequences recognizing surface molecules common to both cell lines, after which the unbound sequences were again incubated with the target cells for positive selection (Table S2). Finally, the ssDNA molecules bound to the target cells were amplified by PCR to be used in the next selection round. A total of 18th rounds of selection were conducted. PCR results from each selection round were monitored by electrophoresis, showing the band for enriched ssDNA pool equal to the initial library with about 90 nucleotides (nt) (data not shown). Flow cytometry was used to monitor the enrichment of the ssDNA pool with binding sequences after the 4th, 5th, 8th, 13th and 17th rounds. The binding ability and specificity of the enriched ssDNA pools were evaluated by incubation with target or control cells and detection of the fluorescence signal. As shown in Fig. 1A, the fluorescence signal with target MDA-MB-231 cells was gradually increased after incubation with the enriched ssDNA pools up to the 8th round. Also, a significant difference between the fluorescence intensity for negative control and target cells after incubation with FAMlabelled ssDNA pools was observed. The results suggest that enriched sequences specific to the target cells were obtained at the 8th round, binding to approximately 20% of the cell population. A decrease of fluorescence signal for the target cells after incubation with the subsequent ssDNA pools was observed, which can be explained by the over-selection that occurs due to PCR artifacts in the amplification steps of cell-SELEX process.
The progression of the selection process was also monitored by measuring the amount of ssDNA retained on cells after washing, obtained after PCR and ssDNA generation (Fig. 1B). These results show an enrichment of ssDNA sequences binding to MDA-MB-231 cells up to the 8th round. Hence, the ssDNA pool from the 8 th round was further sequenced through Illumina MiSeq NGS.

Identification of ssDNA aptamer candidates aided by bioinformatics tools. Sequencing results
after an initial filtration, adapter and constant primer binding region removal and length filtration (49 nt) led   To evaluate the ability of both aptamers to bind to MDA-MB-231 (target) and MCF-10-2A (control) cells, fluorescence microscopy imaging and flow cytometry were performed (Fig. 3C, D). The microscopy and cytometry results are in very good agreement. Apt1 and Apt2 bound to about 75.2 ± 0.4% and 76.2 ± 1.9% of the overall MDA-MB-231 cell population, respectively. Furthermore, it is also shown that for MCF-10-2A, almost no positive signal was detected (1.3 ± 2.2% to Apt1 and 0.2 ± 0.5% to Apt2), thus confirming the specificity of both aptamers. The microscopy results clearly show a bright fluorescence signal for the MDA-MB-231 cell population after incubation with the aptamers, being more pronounced for Apt2. As expected, no fluorescence signal was observed for the control cells, thus corroborating the cytometry results.
Apt1 and Apt2 specifically target cell surface receptors. To investigate whether the incubation temperature could affect the binding ability of both aptamers, MDA-MB-231 cells were incubated with them at different temperatures and then analyzed by flow cytometry. MDA-MB-231 cells showed similar fluorescence intensity patterns after incubation with both aptamers at both temperatures (4 °C and 37 °C), hence demonstrating that the incubation temperature has almost no effect (p > 0.05) on the binding capacity of the selected aptamers (Fig. 4A). In addition, MDA-MB-231 cells, pretreated with trypsin or proteinase K for 3 and 10 min, were incubated with the FAM-labelled aptamers, and were further analyzed by flow cytometry. Both aptamers lost the binding ability to their target cells after treatment with either proteinase K or trypsin at both exposure times (Fig. 4B).
A co-localization experiment was also performed using the naturally fluorescent filipin and both labelled aptamers (Fig. 5A). Apt1 and Apt2 showed a notorious binding to the MDA-MB-231 cell membrane, Table 2. Selected oligonucleotide sequence candidates. Twenty-five most frequent oligonucleotide sequences obtained after next-generation sequencing (NGS) of the 8th Cell-Selex cycle. Only the sequence corresponding to the random region is shown. Primer binding sites were neglected in this representation. www.nature.com/scientificreports/ corroborating the cytometry results. In addition, to delineate cell cytoskeleton, MDA-MB-231 cells were stained with CF 568 Phalloidin and an observable green fluorescence (at a less extent than the observed with filipin due to the great amount of washes during the protocol) was also observed at the membrane level (Fig. S1). In the end, to trace the potential cellular uptake of both aptamers, cells were incubated with lysoSensor ( Fig. 5B). Again, a membrane staining was clearly observed for both aptamers (pointed out by the green arrows) but, interestingly, it was also found some aptamer uptake, represented by the yellow dots (highlighted by the yellow arrows).   Fig. 7, after 24 h of incubation with 250 nM of both aptamers, no significant survival rate change was found, suggesting that both are non-toxic. The same trend was found for Apt2 for a 48 h incubation. However, for Apt1, a significant decrease in cell viability (ρ ≤ 0.05) was observed at this exposure time. The morphological changes resulting from incubation of cells with both aptamers at different time points up to 24 h (non-toxic exposure time for both aptamers) were also studied. As shown in Fig. 7B, no observable differences between the different conditions were found for both aptamers.

Apt1 and
Apt2 has the potential for translational application to clinical oncology. Besides the selected aptamer's ability to recognize MDA-MB-231 cells, we further evaluated their ability to target breast cancer tissues (Fig. 8). For that purpose, fluorescence microscopy was used to image breast tissue sections after incubation with both FAM-labelled aptamers and the library. A visible green fluorescence signal of FAM-labelled Apt2 was detected in human breast tissues and no visible signal was found either for Apt1 or library.

Discussion
Breast cancer is the worldwide leading cause of deaths among women, being TNBC the most aggressive subtype with no approved targeted therapy. The identification of new molecules such as aptamers able to specifically target breast cancer may lead to the development of novel targeted therapies. Herein, cell-SELEX was used to select specific aptamers against the human breast cancer cell line MDA-MB-231, which is a good TNBC model due to its aggressive and metastatic nature. ssDNA aptamers were identified by cell-SELEX against MDA-MB-231 cells and using the normal breast cell line MCF-10-2A for counter-selection to exclude all aptamers that bind in a non-specific way to cancer cells. The enrichment of the ssDNA pool with binding sequences was monitored, being the ssDNA pool from the 8 th round further sequenced by NGS. Afterwards, the sequencing results were filtered and the 25 most frequent oligonucleotide sequences were analysed regarding the sequence alignment and phylogenetic relationship. This type of analysis can provide useful insights on the evolutionary process and on key nucleotides that may be involved in the target binding site 24 . The selected aptamer sequences showed a distinct evolutionary tree, where three main families of related aptamers were identified, meaning that they do not have a similar sequence for conserved nucleotides.
Based on this analysis, it is premature to affirm that these conserved sequences are part of the target binding without confirming whether these regions of interest can contribute to form stems, loops or bulges. All aptamer sequences exhibit a predicted folded-secondary structure with a degree of SL structures. From those, www.nature.com/scientificreports/ two candidate aptamer sequences, Apt1 and Apt2, were chosen based on the sequences' repeatability on the entire pool, but also on their different conserved sequences and secondary structures. A growing number of delivery strategies using cell-specific aptamers as targeting ligands have been proposed 25,26 , given their ability to distinguish small differences in the cell surface protein signature of heterogeneous cancer cells. Therefore, to determine the binding affinity of Apt1 and Apt2 towards MDA-MB-231 cells, their K d values were assessed, giving values of 44.3 ± 13.3 nM and 17.7 ± 2.7 nM, respectively. Various cell-SELEX protocols have been developed to isolate cell-specific aptamers. Li et al. 16 selected the aptamer LXL-1 ( K d = 44 ± 8 nM) against MDA-MB-231 cells. More recently, another approach to select aptamers against circulating tumor MDA-MB-231 cells was attempted. A panel of 5 specific aptamers were identified, from which the M3 aptamer displayed the highest affinity with a K d of 45.6 ± 1.2 nM 19 .
In both abovementioned reports, MDA-MB-231 cell-specific aptamers were identified after Sanger sequencing. NGS revolutionized the aptamer field by increasing the number of reads from few (Sanger sequencing) to millions (HTS methods), thus increasing the probability of finding the best ligands for a given target. The K d values of the aptamers identified in our work are comparable, in the case of Apt1, or with a higher affinity to the target (about 2.5-fold higher), demonstrated for Apt2. A possible explanation is the use of NGS that, as mentioned, enables the retrieval of aptamers with better affinity to the target cells. Even though, this assessment is not straightforward as aptamers were selected using different experimental setups, including different libraries and protocol conditions, making it difficult to do a side by side comparison.
Aptamer selection at other temperatures rather than 37 °C, for instance, 4 °C, may result in poor binding ability at the physiological temperature 27 . However, ssDNA could be non-specifically taken up at physiological www.nature.com/scientificreports/ temperatures by cells through a complex process called endocytosis that occurs via receptor-mediated pathways (clathrin-mediated, caveolae-mediated, macropinocytosis and clathrin and caveolae-independent pathways) 28 . Therefore, MDA-MB-231 cells were incubated with both aptamers at different temperatures, showing similar fluorescence intensity patterns. To preliminary assess whether putative targets of these aptamers are membrane proteins on the cell surface, MDA-MB-231 cells were pre-treated with either trypsin or proteinase K, where both aptamers showed loss of binding ability. These results suggest that the target of both aptamers is associated with epitopes on the cell surface proteins, since proteinase K and trypsin are able to digest extracellular domains of the cell surface without disrupting other components present in the cytosol. Moreover, incubation with filipin showed a clear colocalization with Apt1 and Apt2 at the membrane. Filipin is a naturally fluorescent polyene antibiotic that binds to cholesterol, and because of that, has been widely used as a probe for sterol location in biological membranes 29 .
In addition, to determine if both aptamers have the ability to be internalized upon target membrane binding, MDA-MB-231 cells were incubated with Apt1 or Apt2 and stained with lysoSensor, for fluorescent staining of acidic organelles as lysosomes. A strong accumulation of aptamer on the cell outer margins (pointed out by the green arrows) was again observed, corroborating the abovementioned results. Interestingly, a yellowish signal (highlighted by the yellow arrows) originated inside the cells and co-localized with lysoSensor was also observed. The results suggest that the target molecule of Apt1 and Apt2 is very likely a membrane protein and can be further internalized into cells.
Other authors also identified aptamers that are able to target membrane proteins and afterward are internalized. TLS11a is an aptamer generated by cell-SELEX that exhibit a great affinity against the liver cancer cells 30,31 . Meng et al. 30 demonstrated the internalization ability of TLS11 upon targeting the cell membrane of LH86 cells. These authors generated a selective TLS11a aptamer-doxorubicin (DOX) complex to deliver DOX to liver cancer  33 . Contrarily, MDA-MB-468 was grouped by both research teams as basal A. Finding such aptamers able to stratify such similar TNBC cell lines could be very promising, besides providing insights about novel therapeutic cell-surface targets.
Aptamers need different times to interact with their targets and to produce a cellular response 34 . Furthermore, toxicity is a key limiting factor in the clinical translation and applicability of aptamers. Cell viability was assessed using a standard colimetric experiment that showed that for Apt2 no significant survival rate change was found, independently of the time assayed. However, for Apt1 a significant decrease in cell viability was observed at time point 48 h. The mechanisms underlying the cell viability decrease mediated by Apt1 are not fully understood. Based on these results, we foresee that Apt2 is stable enough for subsequent in vivo studies and possibly will also be useful for future clinical applications.
Aptamers can be used as therapeutic moieties covalently or non-covalently conjugated with a drug to form aptamer-drug conjugates to increase the therapeutic response on target cells 35 . In addition, aptamers can be conjugated to nanoparticles such as liposomes, micelles, polymeric nanoparticles and quantum dots to carry anti-cancer drugs, and to guide the therapeutic reagents to the target cells 35 . Hu et al. 36 exploited the targeting ligand Mucin 1 (MUC1) aptamer for carrying DOX to cancer cells. DOX was intercalated into the DNA aptamer MA3, which binds to MUC1, to generate an aptamer-DOX complex. The complex was able to carry DOX into MUC1 positive tumor cells but lowered the toxicity to MUC1 negative cells. This indicates that the aptamer MA3 has the potential to be used for the targeted delivery of a therapeutic agent to MUC1 expressing tumors 36 . Moreover, there are several aptamers able to internalize cells and inhibit proliferation or induce death. Wang et al. 37   www.nature.com/scientificreports/ and 105.7 nM, respectively 37 . In this sense, our Apt1 could be further explored as a possible cell death inducer due to the significant decrease of cell viability observed. Besides the proved aptamer's ability to recognize MDA-MB-231 cells, the ability to target breast cancer tissues was also evaluated. A notorious green fluorescence signal was found for Apt2, reinforcing and supporting the potential translation of Apt2 to clinical applications, which could be of great significance in TNBC treatment and detection. The ability of aptamers to recognize human cancer tissues has also been demonstrated by other researchers. Wang and co-workers 38 described an aptamer against the receptor glucagon identified by cell-SELEX able to bind to the cell membrane of hepatic tissues. Duan and collaborators 39 identified a DNA aptamer that could be used for imaging clinical metastatic prostate cancer tissues.

Conclusions
In summary, we have successfully demonstrated a combined analysis pipeline to screen aptamers using cell-SELEX aided by a high throughput aptamer identification method to increase the success of aptamers selection against the target MDA-MB-231 cells. The selected aptamers obtained after 8 rounds of evolved enrichment were further aligned and the 25 most frequent oligonucleotide sequences were analyzed. Two aptamer candidates, Apt1 and Apt2, were characterized regarding their binding affinity to the target, with K d values of 44.3 ± 13.3 and 17.7 ± 2.7, respectively. Both aptamers seem to be associated with cell membrane epitopes only present in the target cells and these aptamers can be subsequently internalized. Apt1 and Apt2 toxicity were assessed showing little to no effect on cells, respectively. The potential translation to clinical applications was proven for Apt2 by immunofluorescence staining of tissue sections, thus highlighting its future potential as a ligand for targeted delivery in MDA-MB-231 TNBC tumors.     Cell-SELEX procedure. About 2 × 10 5 cells were plated on 6-well plates two days before the cell-SELEX. At the day of experiment, the culture medium was removed and cells were washed twice with BB and then incubated for 1 min with BlB. The ssDNA library (2 nmol) was prepared in BB, denatured for 5 min at 95 °C, and cooled to down at room temperature for 10 min.

Library, primers and buffers.
For the counter-selection, the initial library was incubated with MCF-10-2A cells for 30 min at 37 °C with gentle shaking. The library solution was collected and centrifuged at 0.3 g for 3 min and the supernatant was used for the selection step using the target MDA-MB-231 cells for 30 min at 37 °C with gentle shaking. After incubation, the supernatant was removed, and cells were washed with WB to remove unbound sequences. Cells were harvested from the well plate. Cells were heated for 10 min at 95 °C in DNAse-free water and cell-bound ssDNA oligonucleotides were amplified by PCR. Round by round the cell-SELEX stringency was augmented by increasing the number of washes after selection and reducing the number of cells for positive selection (Table S2) 40 . About 6.6 µg of purified dsDNA was incubated at 37 °C for 2 h with 25U of lambda exonuclease in 50 µL of total reaction volume. The reaction was stopped by incubation at 75 °C for 10 min. Purified PCR products were analyzed on a 10% denaturing urea-polyacrylamide gel and stained with GelRed [Biotium].
Sequencing and data analysis. The resulting ssDNA pool (from the 8th cycle) with the highest degree of enrichment was sequenced using Illumina MiSeq by Fasteris (Switzerland). The pool for sequencing was prepared by performing an overlap PCR with primers that are complementary to constant regions of the randomized oligonucleotide library with an added overhang that includes an Illumina platform-specific sequence. Afterwards, the sequencing reads were filtered and demultiplexed. For this analysis, constant primer binding regions were removed and sequences longer or shorter than 49 nucleotides were discarded. Raw next-generation sequencing (NGS) data was analyzed using Matlab R2019a (The Mathworks, Inc.) and the alignment algorithm embedded in Geneious 9.1.4 software (Biomatters Ltd.; https:// www. genei ous. com/). The 25 most frequent oligonucleotide sequences were further evaluated. The phylogenetic tree was constructed using Tree Builder function in Geneious, where the tree distances and sequence relatedness was determined by the method of Tamura and Nei using a neighbor-joining model with no outgroup. The free energy (∆G) and secondary structure were calculated using the Mfold software, presenting the optimal structure for each aptamer 23 . The conditions used for the structure predictions were set according to the constituents of the BB at 37 °C in 187 mM Na + and 0.5 mM Mg 2+ at pH 7. In vitro cytotoxicity assay. Cell viability was evaluated using the 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) (MTT) assay [Sigma]. MDA-MB-231 and MCF-10-2A cells (1 × 10 4 cells/well) were plated onto a 96-well plate in the day before the experiment. Cells were incubated for 24 and 48 h at 37 °C with 250 nM of both aptamers, which were previously resuspended in BB, boiled at 95 °C for 5 min, and then cooled for 10 min at room temperature. SB was used as a negative control. After the incubation time, 100 µL MTT solution (0.5 mg/mL) was added to each well and cells were incubated at 37 °C for 4 h. Then, the MTT solution was removed and 150 µL of dimethyl sulfoxide (DMSO) [Sigma] was added to solubilize blue formazan crystals. The absorbance was read at 570 nm in a microplate reader [Cytation 3, BioTek].
Co-localization studies. About 3 × 10 4 cells were seeded on coverslips in 24-well plates and cultured overnight. Cells were incubated with Apt1-F and Apt2-F in BB at 37 °C for 30 min and then were washed twice with WB.
For plasma membrane labelling, about 0.01 mg/ml of filipin [Sigma] dissolved in PBS 1X supplemented with 0.5% BSA was added to cells right before imaging.
For actin filaments staining and cell cytoskeleton delineation, cells were fixed with 4% PFA for 40 min at room temperature. After rinsing with PBS 1X, cells were incubated with 50 mM ammonium chloride [Sigma] for 10 min, rinsed again and permeabilized with 0.1% of sodium dodecyl sulfate (SDS) Sigma) in PBS 1X for 10 min. Next, cells were blocked in PBS 1X supplemented with 3% of BSA for 20 min and washed. Following this procedure, cells were incubated with CF 568 Phalloidin [Biotium] for 1 h in the dark in a humidified atmosphere. After incubation, cells were once again washed with PBS 1X with 0.1% of BSA, DAPI added and incubated for 15 min at room temperature. Coverslips were mounted upside down in Vectashield mounting medium and the images were acquired in a fluorescence microscope [OLYMPUS BX51] incorporated with a high-sensitivity camera Olympus DP71, using a at 60X oil immersion objective.
To trace the cellular uptake of the aptamers, 1 mM of lysoSensor Red DND-99 [ThermoFisher] (1.5 µM final concentration) was added to each sample and incubated during 30 min at 37 °C. Next, the medium was removed and the coverslips were mounted upside down. Images were acquired in a sequential mode by a confocal scanning laser microscope (BX61 FLUOVIEW1000, Olympus), using a 60X oil immersion objective and with the specific filter settings for FAM (laser excitation line 488 nm and emissions filters BA 505-540, green channel) and RED DND-99 dye (laser excitation line 559 nm and emissions filters BA 575-675, red channel).
Fluorescence staining of breast cancer tissue sections. Tumor tissue fluorescence staining was used to confirm the aptamer's ability to bind MDA-MB-231 tissues. Formalin-fixed paraffin-embedded breast cancer tissue sections were deparaffinized, rehydrated and then antigenic retrieval was performed as described elsewhere 41 . Briefly, the slides were heated in 10 mM sodium citrate buffer pH 6.0 [Sigma] at 95 °C for 20 min followed by slow cooling at room temperature for about 20 min. Then, tissue slides were blocked with BlB for Scientific Reports | (2021) 11:8614 | https://doi.org/10.1038/s41598-021-87998-y www.nature.com/scientificreports/ 30 min at room temperature. Afterwards, the slides were incubated with 250 nM of Apt1-F, Apt2-F or SB-F in BB for 30 min at 37 °C and washed with WB. Finally, the slides were stained with Vectashield mounting media containing DAPI solution. Images were acquired using an Olympus BX51 microscope incorporated with a highsensitivity camera Olympus DP71 at 40X magnification.
Statistical analysis. Data were expressed as mean ± standard deviation (SD) of three independent experiments. One-way ANOVA with Tukey's post-test and two-way ANOVA with Sidak's post-test were performed using GraphPad Prism to identify differences among multiple groups, considering a significance level of 95%.